Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 28;23(10):101620.
doi: 10.1016/j.isci.2020.101620. eCollection 2020 Oct 23.

Δ42PD1-TLR4 Augments γδ-T Cell Activation of the Transitional Memory Subset of CD4+ T Cells

Yufei Mo  1 , Allen Ka Loon Cheung  2 , Yue Liu  2 , Li Liu  1 , Zhiwei Chen  1
Affiliations

Δ42PD1-TLR4 Augments γδ-T Cell Activation of the Transitional Memory Subset of CD4+ T Cells

Yufei Mo et al. iScience. .

Abstract

TLR ligands can contribute to T cell immune responses by indirectly stimulating antigen presentation and cytokines and directly serving as co-stimulatory signals. We have previously reported that the human endogenous surface protein, Δ42PD1, is expressed primarily on (Vγ9)Vδ2 cells and can interact with TLR4. Since Vδ2 cells possess antigen presentation capacity, we sought to further characterize if the Δ42PD1-TLR4 interaction has a role in stimulating T cell responses. In this study, we found that stimulation of Vδ2 cells not only upregulated Δ42PD1 expression but also increased MHC class II molecules necessary for the antigen presentation. In a mixed leukocyte reaction assay, upregulation of Δ42PD1 on Vδ2 cells elevated subsequent T cell proliferation. Furthermore, the interaction between Δ42PD1-TLR4 augments Vδ2 cell stimulation of autologous CMV pp65-or TT-specific CD4+ T cell proliferation and IFN-γ responses, which was specifically and significantly reduced by blocking the Δ42PD1-TLR4 interaction. Furthermore, confocal microscopy analysis confirmed the interaction between Δ42PD1+HLA-DR+Vδ2 cells and TLR4+CD4 T cells. Interestingly, the subset of CD4+ T cells expressing TLR4 appears to be PD-1+ CD45RO+CD45RA+ transitional memory T cells and responded to Δ42PD1+HLA-DR+Vδ2 cells. Overall, this study demonstrated an important biological role of Δ42PD1 protein exhibited by Vδ2 antigen-presenting cells in augmenting T cell activation through TLR4, which may serve as an additional co-stimulatory signal.

Keywords: Cell Biology; Immunology.

PubMed Disclaimer

Conflict of interest statement

The antibodies against Δ42PD1 is described in the US patent no. 10,047,137B2 (A.K.L.C. and Z.C.).

Figures

None
Graphical abstract
Figure 1
Figure 1
Δ42PD1 and HLA-DR Expression on Cytokine-Stimulated CD3+Vδ2+ Cells Purified γδ-T cells from healthy PBMCs were isolated and stimulated with different cytokines for 5 days and analyzed for the expression of (A) Δ42PD1, (B) HLA-DR, and (C) co-expression of Δ42PD1/HLA-DR by flow cytometry (n ≥ 8). (D) Representative flow cytometry dot plots of Δ42PD1/HLA-DR co-expression on CD3+Vδ2+ cells, or as column graphs are shown (n = 4) (E). Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S1–S4.
Figure 2
Figure 2
Δ42PD1-TLR4 Augments CD4+ T Cell Stimulation by γδ-T Cells Mixed leukocyte reaction assay between allogeneic PBMCs was performed in the presence of isotype, anti-Δ42PD1, anti-TLR4 antibodies or depleted for γδ-T cells (n = 4). For non-specific antigen stimulation, γδ-T cells were stimulated with γ-irradiated PBMC from an allogeneic donor at a 1:10 ratio, or co-cultured with autologous CFSE-labeled total CD4+ cells at a ratio of 1:1 for 5 days, in the presence of isotype, anti-Δ42PD1, anti-TLR4 antibodies. Transwell was used to separate γ-irradiated PBMC-stimulated γδ-T cells and effector cells. Proliferation was measured by flow cytometry based on CFSE signals. (A and B) (A) Representative CFSE proliferation flow cytometric histograms (numbers indicate percentages), or as (B) column graphs are shown (n = 4). (C) MLR with CFSE-labeled CD4+ T cell proliferation (n ≥ 4). (D) CFSE proliferation of autologous CD4+ T cell proliferation induced by IL-12/IL-15-stimulated γδ-T cells (n = 7). (E and F) (E) γδ-T cells were isolated from CMV-infected PBMCs at day 3 P.I. before being co-cultured (1:5) with pp65 peptide-stimulated autologous T cells to assess intracellular IFN-γ (n = 5), where (F) shows representative overlaid histograms. Neg, unstimulated effector cells; Pos for (A)–(D), PHA/IL-2 stimulation; Pos for (E) PMA/ionomycin. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S5 and S6.
Figure 3
Figure 3
Δ42PD1-TLR4 Interaction between Vδ2 and CD4+ T Cells pp65-Specific CD4+ T cells were used to co-culture with autologous IL-12/IL-15 stimulated and pp65-pulsed γδ-T cells or untreated (Unt) γδ-T cells for around 18 h, before being fixed and immunostained for Δ42PD1 (white), TLR4 (green), HLA-DR (red), and DAPI (blue). Co-culture of pp65-specific CD4+ T cells with (A) unstimulated γδ-T cells or (B) with IL-12/IL-15 stimulated and pp65-pulsed γδ-T cells. Pink and blue arrows point to Δ42PD1+HLA-DR+ and TLR4+ cells, respectively. Pixel size = 120.2 nm. (C) Representative images showing colocalization of Δ42PD1 and HLA-DR expression signals on Unt (n = 65) and pp65-pulsed γδ-T cells (n = 96) were analyzed, for the overlap and Manders' coefficient values shown in the graph. (D) Representative images showing the interaction between pp65-specific CD4+ T cell and pp65-pulsed γδ-T cells analyzed by the signal values for Δ42PD1, MHC-II, and TLR4 along the yellow line transecting from bottom left to top right equivalent to left to right in the histogram. Values are indicated by the histogram of n = 32 cell-cell pairs at ten pixels before and after interface between the cells set at x = 0. (E) Orthogonal views over XY, XZ, and YZ axes of a z-stack of 78 images at 0.29 μm thickness each. Image at stack 72 is shown for the interaction between pp65-specific CD4+ T cell and pp65-pulsed γδ-T cells. Pixel size = 80.2 nm. (F) Imaris analysis on a maximum projection of a z-stack of 25 images each with a thickness of 0.506 μm (top) to generate the 3D model (bottom). Pixel size = 112 nm. The rightmost image is a flipped version of the middle image to indicate interactions at two different angles. White bar represents a scale of 10 μm. Representative images from three independent experiments are shown. Data in the graphs represent mean ± SEM. ∗∗∗p < 0.001. See also Figure S7, Videos S1, S2, and, S3.
Figure 4
Figure 4
TLR4 Is Expressed on CD4+ T Cells PBMC samples were analyzed by flow cytometry for surface TLR4 expression on PD-1+ or PD-1- CD4+ T cells. (A and B) (A) Representative flow cytometry analysis plots on unstimulated PBMCs from n = 21 independent donor samples (B). (C) ECL western blotting detection of TLR4 protein from freshly PBMC-isolated CD4+ T cells, stable expression cell line HEKBlue-hTLR4, and transiently expressed TLR4 on 293T cells. Purified total CD4+ T cells stimulated with anti-CD2/3/28 antibodies were analyzed for TLR4 expression with representative plots shown (D) or as a graph for n = 3 independent donors (E), confirmed by ECL western blotting (F). (G) Detection of TLR4 and β-actin in FACSorted TLR4+ and TLR4- cells after anti-CD2/3/28 activation by Fluorescent western blotting compared with unsorted, unstimulated media control, and control cell lines. Representative blot from one of three donors shown. Ladder markers for (C) and (F) are indicated on the side. (H) Normalized band density (normalizing band density of TLR4 to that of β-actin) was calculated and compared. Column graph data represent mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001. See also Figure S8.
Figure 5
Figure 5
Preferential Response of TLR4+PD-1+CD45RO+CD45RA+ T Cell Subset to Activation by Δ42PD1+Vδ2 cells (A) PBMCs stimulated for 3 days with IL-2, PHA/IL-2, anti-CD2/3/28 antibodies, or left in media, were analyzed by flow cytometry for the frequencies of CD45RO/CD45RA subsets of CD4+ T cell (n ≥ 6). (B) TLR4 expression assessed on PD-1+ or PD-1- subsets of CD45RO/CD45RA CD4+ T cells (n = 6). (C) Intracellular IFN-γ response of CD45RO+CD45RA+CD3+CD4+ T cells stimulated by γδ-T cells treated with IL-12/IL-15/Raji with or without pp65, in the presence of isotype, anti-Δ42PD1, anti-HLA-DR, or anti-PD-L1 antibody. CD4+ T cells treated with PMA/ionomycin (Pos) or media (Neg) served as controls. Four independent experiments were performed. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. See also Figures S9–S13.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ahn E., Araki K., Hashimoto M., Li W., Riley J.L., Cheung J., Sharpe A.H., Freeman G.J., Irving B.A., Ahmed R. Role of PD-1 during effector CD8 T cell differentiation. Proc. Natl. Acad. Sci. U S A. 2018;115:4749–4754. - PMC - PubMed
    1. Allie S.R., Zhang W., Fuse S., Usherwood E.J. Programmed death 1 regulates development of central memory CD8 T cells after acute viral infection. J. Immunol. 2011;186:6280–6286. - PMC - PubMed
    1. Apetoh L., Ghiringhelli F., Tesniere A., Obeid M., Ortiz C., Criollo A., Mignot G., Maiuri M.C., Ullrich E., Saulnier P. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat. Med. 2007;13:1050–1059. - PubMed
    1. Baars P.A., Maurice M.M., Rep M., Hooibrink B., Van Lier R.A. Heterogeneity of the circulating human CD4+ T cell population. Further evidence that the CD4+CD45RA-CD27- T cell subset contains specialized primed T cells. J. Immunol. 1995;154:17–25. - PubMed
    1. Bank I., Cohen L., Mouallem M., Farfel Z., Grossman E., Ben-Nun A. Gammadelta T cell subsets in patients with arthritis and chronic neutropenia. Ann. Rheum. Dis. 2002;61:438–443. - PMC - PubMed